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Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive DrugMetabolites Acting on TRPV1 in the Brain

Barriere, David A.; Mallet, Christophe; Blomgren, Anders; Simonsen, Charlotte; Daulhac,Laurence; Libert, Frederic; Chapuy, Eric; Etienne, Monique; Högestätt, Edward; Zygmunt,Peter; Eschalier, AlainPublished in:PLoS ONE

DOI:10.1371/journal.pone.0070690

2013

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Citation for published version (APA):Barriere, D. A., Mallet, C., Blomgren, A., Simonsen, C., Daulhac, L., Libert, F., ... Eschalier, A. (2013). Fatty AcidAmide Hydrolase-Dependent Generation of Antinociceptive Drug Metabolites Acting on TRPV1 in the Brain.PLoS ONE, 8(8), [e70690]. https://doi.org/10.1371/journal.pone.0070690

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https://doi.org/10.1371/journal.pone.0070690

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https://doi.org/10.1371/journal.pone.0070690

Fatty Acid Amide Hydrolase-Dependent Generation ofAntinociceptive Drug Metabolites Acting on TRPV1 inthe BrainDavid A. Barriere1,2., Christophe Mallet1,2., Anders Blomgren3, Charlotte Simonsen3,

Laurence Daulhac1,2, Frederic Libert1,2, Eric Chapuy1,2, Monique Etienne1,2, Edward D. Hogestatt3*,

Peter M. Zygmunt3, Alain Eschalier1,2,4*

1 Clermont Universite, Universite d’Auvergne, Pharmacologie Fondamentale et Clinique de la Douleur, Laboratoire de Pharmacologie, Facultes de Medecine/Pharmacie,

Clermont-Ferrand, France, 2 Inserm, U1107 Neuro-Dol, Clermont-Ferrand, France, 3 Department of Clinical Chemistry and Pharmacology, Lund University, Lund, Sweden,

4 CHU Clermont-Ferrand, Service de Pharmacology, Hopital G. Montpied, Clermont-Ferrand, France

Abstract

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents viaactivation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics.Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamolmetabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amidehydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide(HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1activators was arvanil = olvanil..AM404. HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity invarious rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in themouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of theTRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test.In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, andchemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, thepharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting oursuggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation of bulbospinal pathways.Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as ametabolic pathway for the generation of TRPV1 modulators in the CNS.

Citation: Barriere DA, Mallet C, Blomgren A, Simonsen C, Daulhac L, et al. (2013) Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive DrugMetabolites Acting on TRPV1 in the Brain. PLoS ONE 8(8): e70690. doi:10.1371/journal.pone.0070690

Editor: Silvana Gaetani, Sapienza University of Rome, Italy

Received March 6, 2013; Accepted June 21, 2013; Published August 5, 2013

Copyright: � 2013 Barriere et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the ‘‘Association Nationale de la Recherche Technique’’, Bristol-Myers-Squibb (grants to CM and DAB), the University ofAuvergne, Inserm, the Swedish Medical Research Council (2010–3347) and the Medical Faculty of Lund. The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.

Competing Interests: CM and DAB have received funding from Bristol-Myers-Squibb. However, this does not alter the authors’ adherence to all the PLOS ONEpolicies on sharing data and materials.

* E-mail: [email protected] (AE); [email protected] (EDH)

. These authors contributed equally to this work.

Introduction

Paracetamol is an effective analgesic, a single oral dose of

1000 mg having an NNT (numbers needed to treat) of 3.6 for at

least 50% pain reduction over 4–6 hours in patients with

postoperative pain [1]. However, the formation of toxic metab-

olites, such as N-acetyl-p-benzoquinone imine (NAPQI), in the

liver is becoming an increasing concern particularly during long-

term use, potentially preventing the full exploitation of paracet-

amol’s analgesic activity. We have previously demonstrated that

paracetamol is metabolized to the TRPV1 activator AM404 and

that activation of TRPV1 in the brain by this metabolite

contributes to the antinociceptive activity of oral paracetamol in

rodents [2–4]. Mapping the in vivo metabolism of paracetamol in

different organs indicated an extensive de-acetylation to the

primary amine 4-aminophenol in the liver [2]. This well-known

paracetamol metabolite enters the brain, where it undergoes

conjugation with arachidonic acid to yield AM404, a reaction

dependent on the enzyme FAAH [2]. The expression pattern in

adult animals and the close evolutionary development of TRPV1

and FAAH implicate a functional relationship between these

proteins in the nociceptive pathways [5–8]. Thus, the design of

molecules undergoing FAAH-dependent biotransformation to

TRPV1 active drug metabolites in the CNS represents a plausible

PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e70690

strategy for developing novel antinociceptive agents that may be

more effective and less toxic than paracetamol.

Here we validated this strategy by examining whether systemic

administration of the primary amines 4-aminophenol, the de-

acetylated metabolite of paracetamol, and HMBA, none of which

are directly oxidized to NAPQI, can produce antinociception via

the generation of TRPV1 active drug metabolites in the brain.

Using mass spectrometry and various rodent nociceptive tests, we

show that both 4-aminophenol and HMBA produce antinocicep-

tion and undergo fatty acid conjugation in the brain, leading to the

formation of AM404 plus HPODA and arvanil plus olvanil,

respectively, of which arvanil and olvanil are extremely powerful

TRPV1 activators with potencies in the subnanomolar range [9–

11]. As shown for paracetamol, the antinociceptive effects of 4-

aminophenol were dependent on FAAH, TRPV1, cannabinoid

CB1 receptors and serotonergic mechanisms [3,12], supporting

our previous suggestion that 4-aminophenol is a key intermediate

metabolite in the bioactivation of paracetamol.

Results

FAAH-dependent Metabolism of 4-aminophenol andHMBA

The primary amine 4-aminophenol is an excellent substrate for

FAAH in the biosynthesis of AM404 in brain homogenates from

mice and rats [2]. Here we show that incubation of mouse brain

homogenates with another primary amine, HMBA, leads to the

formation of arvanil and olvanil (Fig. 1A), which are both

recognized as potent TRPV1 activators [9–11]. To establish

whether fatty acid conjugation of 4-aminophenol and HMBA also

occurs in living animals, we determined the contents of AM404,

HPODA, arvanil and olvanil in the brain 20 min after intraper-

itoneal injection of 4-aminophenol (30 and 100 mg/kg) and

HMBA (100 and 300 mg/kg) in mice. These N-acylamines were

all found to be potent TRPV1 activators (Fig. 1B), arvanil

(pEC50 = 9.760.1, n = 12) and olvanil (pEC50 = 9.560.2, n = 7)

being 172 and 110 times more potent than AM404

(pEC50 = 7.560.1, n = 6), respectively, whereas AM404 was 3.3

times more potent than HPODA (pEC50 = 7.060.1, n = 9), using

capsazepine-sensitive vasorelaxation as readout (Fig. S1). AM404

and HPODA as well as arvanil and olvanil were detected after

administration of both doses of 4-aminophenol and HMBA,

respectively (Table 1 and 2), but not after vehicle injection (n = 6).

However, olvanil only surpassed the level of quantification after

the high dose of HMBA (300 mg/kg).

We also determined the contents of 4-aminophenol and HMBA

in blood and the brain from the same animals (Table 1 and 2). A

conspicuous difference between 4-aminophenol and HMBA was

observed in the quotient between the brain levels of these

molecules and their lipid metabolites, a parameter most likely

reflecting the efficiency of the conjugation reaction. The quotients

between 4-aminophenol and AM404 (HPODA) were 223 (951)

and 1,547 (9,376) for the 30 mg/kg and 100 mg/kg doses of 4-

aminophenol, respectively. The corresponding quotients between

HMBA and arvanil (olvanil) were 39,773 (not determined) and

69,980 (86,307) for the 100 mg/kg and 300 mg/kg doses of

HMBA, respectively. At an identical dose of 4-aminophenol and

HMBA (100 mg/kg), the brain content of AM404 was approx-

imately three orders of magnitude higher than that of arvanil

(Table 1 and 2). Furthermore, the quotient between the contents

of AM404 and 4-aminophenol (1:1,500) was substantially larger

than the quotient between the contents of arvanil and HMBA

(1:40,000), again indicating a more efficient conversion of 4-

Figure 1. Production of TRPV1 active drug metabolites in themouse brain from 4-aminophenol and 4-hydroxy-3-methox-ybenzylamine (HMBA). Formation of arvanil and olvanil in rat brainhomogenates incubated with HMBA (100 mM) for 60 min (A; n = 6).These N-acylamines were not detected in brain homogenates exposedto vehicle only. TRPV1-dependent vasorelaxation evoked by AM404(n = 6), N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA; n = 9), arvanil(n = 12) and olvanil (n = 7) in rat mesenteric arterial segmentsprecontracted with phenylephrine (B). Content of AM404 (C and E)and HPODA (D and F) in the brain 20 min after intraperitoneal injectionof 4-aminophenol at doses of 30 mg/kg (C and D; n = 5) and 100 mg/kg(E; n = 6–12 and F; n = 6) in different FAAH genotypes. Contents of

Analgesic TRPV1 Active Drug Metabolites in Brain


aminophenol to AM404 than of HMBA to arvanil in the mouse

brain.

We have previously demonstrated that the formation of AM404

in the mouse brain is dependent on FAAH after systemic

administration of paracetamol [2]. Here we show that the brain

contents of AM404, arvanil and olvanil, but not HPODA, were

substantially reduced in FAAH2/2 mice compared to their

FAAH+/+ littermates 20 min after intraperitoneal injection of 4-

aminophenol (30 and 100 mg/kg; Fig. 1C and E) and HMBA

(300 mg/kg; Fig. 1G and H). We could not extend these findings

to the low dose of HMBA (100 mg/kg), because the brain contents

of olvanil and arvanil were either below or immediately above the

level of quantification in wild-type mice, respectively. Notably, the

brain content of HPODA increased 3-fold in FAAH2/2 mice

compared to their wild-type littermates after the low dose of 4-

aminophenol, while no significant difference was found between

the genotypes after the high dose of 4-aminophenol (Fig. 1D and

F).

The content of AM404 in brain was also determined at different

time points after intraperitoneal administration of 4-aminophenol

(100 mg/kg), as shown in the rat. Significant amounts of AM404

were detected 15 min and 30 min after 4-aminophenol injection

(Fig. 2A). Pretreatment of the animals with the FAAH inhibitor

phenylmethanesulfonyl fluoride (PMSF; 10 mg/kg s.c.) substan-

tially reduced the formation of AM404, as measured 20 min after

4-aminophenol administration (Fig. 2B). These findings show that

the in vivo condensation of 4-aminophenol and arachidonic acid is

dependent on FAAH and support our previous notion that 4-

aminophenol is a key intermediate metabolite in the bioactivation

of paracetamol in the rodent brain [2,3,12].

FAAH- and TRPV1-dependent Antinociceptive Effects of4-aminophenol and HMBA

We first tested the effects of 4-aminophenol and HMBA on

locomotor activity, a potential bias in the assessment of

antinociceptive activity. Locomotor activity was assessed 10 min

after an intraperitoneal injection of 4-aminophenol (10, 30 and

100 mg/kg in mice) and HMBA (100, 200 and 300 mg/kg in

mice) or vehicle. Mice showed reduced locomotor activity after the

highest dose of 4-aminophenol (Fig. 3A), whereas rats tolerated all

three doses (data not shown). For HMBA, only the dose of

100 mg/kg failed to affect locomotion (Fig. 3B).

Involvement of FAAH. As demonstrated in FAAH+/+ mice,

4-aminophenol at a dose of 30 mg/kg (i.p.) reduced the licking

time during the first and second phases of the formalin test,

increased the withdrawal threshold in the von Frey test and the

latency time in the tail immersion test, indicating a clear

antinociceptive activity (Fig. 4A–D). If 4-aminophenol is a key

intermediate in the bioactivation of paracetamol, the antinocicep-

tive effect of 4-aminophenol should be dependent on FAAH.

Indeed, this was found to be the case, as 4-aminophenol failed to

produce antinociception in all three pain tests in FAAH2/2 mice

(Fig. 4B–D).

In the rat, the effects of three doses of 4-aminophenol (10, 30

and 100 mg/kg i.p.) were examined in the formalin test. Only the

highest dose reduced both phases of the formalin test, as measured

by the licking time (Fig. 5A). This dose of 4-aminophenol also

increased the withdrawal threshold in the rat paw pressure test 15

and 30 min after drug administration, while the intermediate dose

(30 mg/kg) was effective only at 15 min and the low dose (10 mg/

kg) was inactive in this test of acute mechanical nociception

(Fig. 5B).

We have previously reported that pretreatment with the FAAH

inhibitor PMSF (10 mg/kg s.c.) prevents the antinociceptive

activity of paracetamol in the rat [12]. Here we show that PMSF

also inhibited the antinociceptive effect of 4-aminophenol

(100 mg/kg i.p.) in the rat formalin and paw pressure tests

(Fig. 5C and D). Although PMSF may inhibit several serine

proteases, these findings are consistent with 4-aminophenol being

a key intermediate metabolite contributing to the antinociceptive

action of paracetamol.

We further examined the effect of the primary amine HMBA in

the mouse formalin test. As this drug is metabolized to the

ultrapotent TRPV1 activators arvanil and olvanil in the rodent

brain, we expected it to possess antinociceptive activity similar to

that of paracetamol or 4-aminophenol. HMBA (100 mg/kg)

inhibited both the first and second phases of the formalin test in

wild-type mice (Fig. 6A), but affected none of these phases in

FAAH2/2 mice in contrast to their wild-type littermates (Fig. 6B).

It was recently shown that the analgesic dipyrone is also

subjected to a FAAH-dependent metabolic conversion to bioactive

N-arachidonoylamines that accumulate in the mouse CNS after

repeated administration [13]. One of these metabolites behaved as

a weak blocker of TRPV1-mediated calcium responses in vitro with

an IC50 of approximately 3 mM [14]. We found that dipyrone

(50 mg/kg i.p.) is an effective antinociceptive agent in the mouse

formalin test and that this action is independent of FAAH, as the

compound produced similar effects in FAAH2/2 mice and their

wild-type littermates (Fig. 6C).

Involvement of TRPV1. We have previously reported that

genetic inactivation of TRPV1 abolishes the antinociceptive effects

of paracetamol in the mouse formalin, von Frey and tail

immersion tests [3]. Here we show that these findings extend to

arvanil (G; n = 6) and olvanil (H; n = 6) in the brain 20 min afterintraperitoneal injection of HMBA at a dose of 300 mg/kg in differentFAAH genotypes. Values are expressed as mean 6 SE. *p,0.05,**p,0.01 and ***p,0.001 when compared to wild-type littermates,using Mann-Whitney U test (A, C, D, G and H) or Kruskal-Wallis one-wayANOVA followed by Dunn’s multiple comparisons test (E and F).doi:10.1371/journal.pone.0070690.g001

Table 1. Quantification of 4-AP, AM404 and HPODA 20 min after 4-AP injection (i.p.).

Blood Brain

4-AP (dose)# 4-AP (nmol/g) 4-AP (nmol/g) AM404 (pmol/g) HPODA (pmol/g)

30 mg/kg 283615 91612 4116134 96616

100 mg/kg 8786157 747061720 482961435 7976114

4-AP, 4-aminophenol; HPODA, N-(4-hydroxyphenyl)-9Z-octadecenamide; i.p., intraperitoneal.#n = 5-6 mice.doi:10.1371/journal.pone.0070690.t001



4-aminophenol (30 mg/kg i.p.), the antinociceptive activity of

which is also lost in TRPV12/2 mice in these tests (Fig. 7A–C).

Furthermore, pretreatment of rats with the TRPV1 blocker

capsazepine (10 mg/kg i.p.) prevented the antinociceptive effect of

4-aminophenol in the formalin and paw pressure tests (Fig. 8A and

B).

These strategies to inactivate TRPV1 do not address the site of

action of a drug given systemically. Therefore, to selectively target

TRPV1 in the CNS, capsazepine (100 nmol) was injected into the

lateral ventricle 5 min prior to 4-aminophenol (30 mg/kg i.p.) or

HMBA (100 mg/kg i.p.) administration in mice. Capsazepine

eliminated the antinociceptive effects of 4-aminophenol and

HMBA in the mouse formalin test, rendering the drugs inactive

on both phases of the test (Fig. 9A and B). Inspection of the brain

and the thoracic and lumbar spinal cord after methylene blue

injection demonstrated that staining was confined to brain tissue

surrounding the cerebral ventricles (n = 4).

Further Evidence for a Similar Pharmacological Profile of4-aminophenol and Paracetamol

Involvement of cannabinoid CB1 receptors. It is intrigu-

ing that the antinociceptive effect of paracetamol in, e.g., the rat

formalin and paw pressure tests is inhibited by the cannabinoid

CB1 receptor antagonist AM251 [12]. Again, we find that

observations made on paracetamol can be extended to 4-

aminophenol, as pretreatment with the cannabinoid CB1 receptor

antagonist AM251 (3 mg/kg i.p.) prevented the antinociceptive

effect of 4-aminophenol (100 mg i.p.) in the rat formalin and paw

pressure tests (Fig. 10A and B). Notably, this dose of 4-

aminophenol did not affect the global levels of the endocannabi-

noids anandamide and 2-arachidonoylglycerol in the rat brain 15,

30, 45 and 60 min after 4-aminophenol administration (Fig. 10C

and D), as previously shown for paracetamol [2].

Involvement of spinal serotonergic mechanisms. Studies

in both animals and man have implicated an important role of

bulbospinal pathways and serotonergic mechanisms in the

analgesic effect of paracetamol [12,15–17]. We therefore explored

whether spinal serotonergic mechanisms are also involved in the

antinociceptive effect of 4-aminophenol. The neurotoxin 5,7-

dihydroxytryptamine (5,7-DHT) was injected intrathecally

(100 mg/rat) to produce lesion of spinal serotonergic pathways in

the rat. This treatment reduced the spinal serotonin content from

529634 to 359638 ng/g tissue wet weight (p,0.05) and inhibited

the antinociceptive effect of 4-aminophenol, as measured 7 days

after 5,7-DHT treatment (Fig. 11A). Furthermore, intrathecal

injection of the 5-HT3 receptor antagonist tropisetron (500 ng/rat)

and the selective 5-HT1A receptor antagonist WAY-100635

(40 mg/rat) 5 min before administration of 4-aminophenol

(100 mg/kg i.p.) prevented, as for paracetamol [12], the antino-

ciceptive effect in the rat paw pressure and formalin tests,

respectively (Fig. 11B and C).

Table 2. Quantification of HMBA, arvanil and olvanil 20 min after HMBA injection (i.p.).

Blood Brain

HMBA (dose)# HMBA (nmol/g) HMBA (nmol/g) Arvanil (pmol/g) Olvanil (pmol/g)

100 mg/kg 220654 132673 3.3360.33 ,LoQ*

300 mg/kg 2206667 38956284 56610 4565.6

HMBA, 4-hydroxy-3-methoxybenzylamine; i.p., intraperitoneal.#n = 6 mice.*Below the level of quantification (LoQ).doi:10.1371/journal.pone.0070690.t002

Figure 2. Content of AM404 in the rat brain after intraperito-neal injection of 4-aminophenol (100 mg/kg). Brain content ofAM404 at different time points after 4-aminophenol administration (A;n = 6). Brain content of AM404 20 min after 4-aminophenol injection inanimals pretreated with phenylmethanesulfonyl fluoride (PMSF; 10 mg/kg s.c.) 20 min before 4-aminophenol administration (B; n = 6). TheAM404 content is presented as normalized peak area (nPA) per gprotein. Results are expressed as mean 6 SE. **p,0.01 and ***p,0.001when compared to time zero (A) or vehicle-treated animals (B), usingKruskal-Wallis one-way ANOVA followed by Dunn’s multiple compari-sons test (A) or Mann-Whitney U test (B).doi:10.1371/journal.pone.0070690.g002

Figure 3. Effects of 4-aminophenol and 4-hydroxy-3-methox-ybenzylamine (HMBA) on spontaneous locomotor activity inthe mouse. Locomotor activity was assayed by counting the numberof crossings of light beams in actimetry boxes over a 15 min period.Doses of 4-aminophenol (A; n = 4) and HMBA (B; n = 6) below 100 mg/kg and 200 m/kg, respectively, failed to impair locomotor activity. 4-Aminophenol and HMBA were injected by the intraperitoneal route10 min before the test. *p,0.05, **p,0.01 and ***p,0.001 whencompared to vehicle injection, using Kruskal-Wallis one-way ANOVAfollowed by Dunn’s multiple comparisons test.doi:10.1371/journal.pone.0070690.g003



Discussion

The staggering literature on paracetamol provides a complex

picture with several metabolites and mechanisms potentially

contributing to paracetamols analgesic activity, including TRPV1

activation in the brain by AM404 and possibly other N-4-

(hydroxyphenyl)acylamide metabolites of paracetamol [2,3],

recruitment of bulbospinal serotonergic pathways [12,15–17],

activation of spinal TRPA1 by electrophilic paracetamol metab-

olites [18] and inhibition of prostaglandin production [19]. In the

present study, we show that systemic administration of 4-

aminophenol and HMBA generates potent TRPV1 active drug

metabolites in the mouse brain and produces antinociception in

various pain tests, including the mouse formalin (4-aminophenol,

HMBA), von Frey (4-aminophenol) and tail immersion (4-

aminophenol) tests, and the rat formalin (4-aminophenol) and

paw pressure (4-aminophenol) tests. The brain levels of the

TRPV1 active drug metabolites, except HPODA, were substan-

tially reduced in FAAH2/2 mice and the antinociceptive activities

of 4-aminophenol and HMBA disappeared in FAAH2/2 and

TRPV12/2 mice. Similar results were obtained in rats subjected

to pharmacological inhibition of FAAH and TRPV1. It is unlikely

that inhibition of prostaglandin synthesis contributed to the

antinociceptive effects of 4-aminophenol and HMBA, because

the mouse tests used, except for the second phase of the formalin

test, reflect non-inflammatory acute pain that is independent of

COX [3]. These findings support our proposal that activation of

TRPV1 by FAAH-dependent drug metabolites underlies the

antinociceptive activity of 4-aminophenol and HMBA in these

tests, as has been shown for paracetamol (Fig. 12) [3]. The

similarities between the antinociceptive activities of 4-aminophe-

nol and paracetamol in terms of their dependence on FAAH,

TRPV1, cannabinoid CB1 receptors and spinal serotonergic

mechanisms provide additional evidence that 4-aminophenol is

the key intermediate metabolite in the bioactivation of paracet-

amol.

We show that 4-aminophenol and HMBA given systemically

produced a dose-dependent formation of AM404 plus HPODA

and arvanil plus olvanil in the mouse brain, respectively, and that

intracerebroventricular injection of the TRPV1 blocker capsaze-

pine prevented the antinociceptive effects of 4-aminophenol and

HMBA in the mouse formalin test. We could not detect any

staining of the thoracic and lumbar spinal cord after intracer-

ebroventricular injection of the water soluble dye methylene blue.

Thus, it is unlikely that capsazepine directly interfered with the

primary afferent input to the dorsal horn of the spinal cord

following formalin injection. However, we cannot exclude an

interaction with TRPV1 on central projections of primary

afferents terminating in the trigeminal or solitary tract nuclei.

Taken together, our findings indicate that activation of TRPV1 in

brain plays a key role in the antinociceptive effects of 4-

aminophenol and HMBA and suggest that lipid metabolites, such

as AM404 and arvanil, mediate these actions.

As shown previously, intracerebroventricular injection of

AM404 can reproduce the TRPV1-dependent antinociceptive

effect of oral paracetamol in the mouse formalin test [3], although

the duration of action of AM404 was relatively short, probably

reflecting rapid elimination of AM404 in the absence of its

precursor 4-aminophenol [20]. Arvanil given by the same route

also possesses antinociceptive activity, as shown in the mouse tail-

flick test [21]. However, the brain contents of arvanil (3.33 pmol/

g) and olvanil (below the level of quantification) were much lower

than the content of AM404 (411 pmol/g) and HPODA (96 pmol/

g) after systemic administration of HMBA (100 mg/kg) and 4-

aminophenol (30 mg/kg), respectively. This does not necessarily

mean that arvanil and olvanil produced less TRPV1 activation

than AM404 and HPODA, because the final responses also

depend on the potencies of the compounds. Interestingly, arvanil

was shown to be equipotent with the ultrapotent TRPV1 activator

resiniferatoxin in functional assays and radioligand binding studies

of rodent TRPV1 [9,11]. We have identified two previous studies

that directly compare the TRPV1 activity of AM404 with that of

arvanil or olvanil. One study reported that arvanil was 933 times

Figure 4. The antinociceptive effect of 4-aminophenol isdependent on fatty acid amide hydrolase (FAAH) in mouse.The antinociceptive effect of 4-aminophenol, given at a dose that didnot affect spontaneous locomotor activity (Fig. 3), was lost in FAAH2/2

mice as compared to their wild-type littermates. The effect of 4-aminophenol at a dose of 30 mg/kg (i.p.) was assessed in the formalintest in C56BL/6 wild-type mice (A; n = 8) and in the formalin (B; n = 6–9),von Frey (C; n = 6) and tail immersion (D; n = 6) tests in FAAH2/2 miceand their wild-type littermates. 4-Aminophenol was injected by theintraperitoneal route 10 min before the tests. *p,0.05, **p,0.01 and***p,0.001 when compared to vehicle injection, using Mann-WhitneyU test.doi:10.1371/journal.pone.0070690.g004



more potent than AM404 in a 45Ca2+ uptake assay, using CHO

cells expressing the rat orthologue of TRPV1 [11]. In another

study, arvanil and olvanil were approximately 50 times more

potent than AM404 in HEK293 cells expressing the human

TRPV1, using fluorometric calcium imaging [10]. In the present

study, we found that arvanil and olvanil were .100 times more

potent than AM404 and .300 times more potent than HPODA

as TRPV1 activators. Taking these potency differences into

account, it is likely that the amounts of AM404 plus HPODA and

arvanil plus olvanil formed after 4-aminophenol (30 mg/kg) and

HMBA (100 mg/kg) administration, respectively, produced com-

parable TRPV1 activation in the mouse brain. Indeed, these doses

of HMBA and 4-aminophenol produced equivalent antinocicep-

tion in the mouse formalin test.

The contribution of HPODA to the antinociceptive effect of 4-

AP is, however, less clear. This N-acylamine was not only less

potent than AM404 as a TRPV1 activator, but its level in the

mouse brain after 4-aminophenol administration was approxi-

mately one fourth of that of AM404. More importantly, while the

antinociceptive activity of 4-aminophenol (30 mg/kg) disappeared

in FAAH2/2 mice, the brain content of HPODA after this dose of

4-aminophenol was 3 times larger in these mice than in their wild-

type littermates. Thus, in contrast to the oleic acid derivative of

HMBA (olvanil), the corresponding derivative of 4-aminophenol

(HPODA) seems to be generated mainly by a FAAH-independent

biosynthetic pathway, although FAAH may mediate the degrada-

tion of both these N-acylamines. These findings indicate not only

quantitative differences, but also qualitative differences in the fatty

acid conjugation of 4-aminophenol and HMBA.

AM404 was originally introduced as a selective inhibitor of

cellular anandamide uptake and degradation [22–24]. However,

the potency of AM404 as an inhibitor of anandamide degradation

in rat brain homogenates is between two and three orders of

magnitude lower than the potency of this compound in assays of

TRPV1 activity [2,10,11]. The selectivity towards TRPV1 is even

larger for arvanil and olvanil, which both are weak inhibitors of

anandamide uptake and degradation [10,23,25–27]. This is in line

with the global brain levels of endocannabinoids being unaffected

by systemic administration of 4-aminophenol (present study) or

paracetamol [3]. It is therefore intriguing that the cannabinoid

CB1 receptor antagonist AM251 could prevent the antinociceptive

effect of 4-aminophenol in the rat formalin and paw pressure tests.

We have previously reported similar findings with respect to

paracetamol [12]. Unsaturated long chain N-acyl-hydroxypheny-

lamides and -vanillylamides, including AM404, arvanil and

olvanil, are poor cannabinoid receptor agonists [21,24–27]. It is,

Figure 5. The antinociceptive effect of 4-aminophenol is dependent on fatty acid amide hydrolase (FAAH) in rats. 4-Aminophenol at adose of 100 mg/kg significantly reduced the first and second phases of the formalin test (A; n = 7–8). Both the 30 mg/kg and the 100 mg/kg doses of4-aminophenol increased the withdrawal threshold in the paw pressure test (B; n = 8). Pre-treatment with phenylmethanesulfonyl fluoride (PMSF;10 mg/kg) substantially reduced or prevented the antinociceptive effect of 4-aminophenol (100 mg/kg) in the formalin (C; n = 10–14) and pawpressure (D; n = 7–11) tests. The different doses of 4-aminophenol were injected by the intraperitoneal route 10 min before the tests. PMSF wasinjected subcutaneously 20 min before 4-aminophenol administration. *p,0.05, **p,0.01 and ***p,0.001 when compared to vehicle injection,using Kruskal-Wallis one-way ANOVA followed by Dunn’s test (A), repeated measures two-way ANOVA followed by Dunnetts (B) or Sidak’s (D)multiple comparisons tests, or Mann-Whitney U test (C).doi:10.1371/journal.pone.0070690.g005



thus, unlikely that sufficient amounts of AM404 and arvanil plus

olvanil are generated from 4-aminophenol and HMBA in the

brain, respectively, to sustain a direct activation of the cannabinoid

CB1 receptor. A recent study, addressing the intriguing antino-

ciceptive effect of the TRPV1 activator capsaicin in the

periaqueductal gray (PAG), an important midbrain region for

regulation of descending pain inhibitory pathways projecting to

the dorsal horn [28,29], has provided evidence of the recruitment

of cannabinoid CB1 receptors downstream of TRPV1 present on

excitatory glutamatergic neurons in the ventrolateral PAG [30].

Microinjection of capsaicin into the rat ventrolateral PAG reduced

the nocifensive behaviour in the hot plate test, an effect that was

inhibited by co-injection of capsaicin with the cannabinoid CB1

receptor antagonist AM251 as well as with the selective TRPV1

blocker SB 366791 [30]. Patch-clamp recordings in brain slices of

the ventrolateral PAG demonstrated that capsaicin produced not

only a glutamate-dependent neuronal excitation, but also an

endocannabinoid-mediated retrograde inhibition of GABAergic

neurons, an effect reversed by AM251 [30]. Such a dual effect of

TRPV1 activators in the ventrolateral PAG may explain the

seemingly contradictory finding that pharmacological or genetic

inactivation of the cannabinoid CB1 receptor is able to inhibit the

antinociceptive effects 4-aminophenol and paracetamol.

Bulbospinal serotonergic pathways originate in several brain-

stem nuclei, including the nucleus raphe magnus. These pathways

receive input from PAG and contribute to the descending

regulation of nociceptive signalling via serotonin release and

activation of its cognate receptors on dorsal horn neurons [31].

Our findings that intervention with spinal serotoninergic mecha-

nisms prevents the antinociceptive effects of 4-aminophenol

(present study) and paracetamol [12] provide additional evidence

that supraspinal TRPV1 may regulate nociceptive neurotransmis-

sion via activation of descending bulbospinal pathways [32].

TRPV1 activation also facilitates glutamate neurotransmission in

the dorsolateral PAG and in other brain areas of potential

importance for the regulation of descending pain inhibition,

including the locus coeruleus and paraventricular nucleus [33–36].

Thus, the identity and localization of the TRPV1-expressing

Figure 6. Effects of 4-hydroxy-3-methoxybenzylamine (HMBA)on nociception in the formalin test in mice. At a dose that did notaffect spontaneous locomotion (Fig. 3B), HMBA (100 mg/kg) inhibitednocifensive behaviour in both phases of the formalin test in C57BL/6 wild-type mice (A; n = 5–7). The antinociceptive effect of HMBAdisappeared in FAAH2/2 mice as compared to their wild-typelittermates (B; n = 4–8), whereas dipyrone (30 mg/kg) produced similarantinociception in FAAH+/+ and FAAH2/2 mice (C; n = 5–7). HMBA anddipyrone were injected by the intraperitoneal route 10 min before thetests. *p,0.05 and **p,0.01 when compared to vehicle injection, usingMann-Whitney U test.doi:10.1371/journal.pone.0070690.g006

Figure 7. The antinociceptive effect of 4-aminophenol isdependent on TRPV1 in the mouse. The antinociceptive effect of4-aminophenol (30 mg/kg) in the mouse formalin (A; n = 5–6), von Frey(B; n = 6) and tail immersion (C; n = 6) tests disappeared in TRPV12/2

mice as compared to their wild-type littermates. 4-Aminophenol wasinjected by the intraperitoneal route 10 min before the tests. **p,0.01when compared to vehicle injection, using Mann-Whitney U test.doi:10.1371/journal.pone.0070690.g007



neurons activated by the lipids metabolites of paracetamol, 4-

aminophenol and HMBA, and the relationship between these

neurons and the descending bulbospinal serotonergic pathways

remain to be elucidated.

Several factors may influence the biotransformation of 4-

aminophenol and HMBA to TRPV1 active lipid metabolites in

the brain, including drug absorption from the site of injection,

distribution of the drug to the brain and FAAH-dependent fatty

acid conjugation to yield the various bioactive metabolites. Our

pharmacokinetic findings show that relatively small changes in the

chemical structure may have profound effects on the propensity of

the molecule to undergo fatty acid conjugation. Indeed, mice

receiving equieffective doses of 4-aminophenol (30 mg/kg) and

HMBA (100 mg/kg) in the mouse formalin test produced similar

brain contents of 4-aminophenol (91 nmol/g) and HMBA

(132 nmol/g). However, the corresponding brain contents of

AM404 and arvanil were 411 pmol/l and 3.33 pmol/l, respec-

tively, indicating that the FAAH-mediated conjugation with

arachidonic acid was approximately 100 times more efficient for

4-aminophenol than HMBA, two structurally similar primary

amines (Fig. 12).

The hepatotoxicity of 4-aminophenol is very low in rodents and

may involve its N-acetylation to paracetamol [37,38]. However, 4-

aminophenol has been shown to produce renal tubular necrosis

after a single exposure at doses not far from those eliciting

antinociception in rodents [39–41]. This selective nephrotoxic

effect seems to depend on the extrarenal formation of toxic

glutathione conjugates of 4-aminophenol, including 4-amino-3-

cysteinylphenol, which then accumulates in proximal tubular

epithelial cells, although a direct cytotoxic effect of 4-aminophenol

on tubular epithelial cells cannot be excluded [42,43]. The

glutathione transferase capacity differs greatly between species and

it is particularly high in rats and mice, making predictions of

tolerable doses in man very difficult [44]. In contrast to 4-

aminophenol and paracetamol, HMBA should not generate

reactive benzoquinone imines and therefore may display low

nephro- and hepatotoxicity. Further animal studies addressing the

toxicology and the antinociceptive activity in models of inflam-

matory pain are warranted before considering 4-aminophenol and

HMBA as potential analgesics for parenteral use in man.

This study suggests that FAAH-dependent generation of

antinociceptive TRPV1 active drug metabolites in the brain

represent a novel pharmacological principle for treatment of pain

and strengthens that 4-aminophenol is the key FAAH substrate in

the bioactivation of paracetamol.

Figure 8. The antinociceptive effect of 4-aminophenol isdependent on TRPV1 in the rat. The TRPV1 blocker capsazepine(10 mg/kg) prevented the antinociceptive effect of 4-aminophenol(100 mg/kg) in the rat formalin (A; n = 7–10) and paw pressure (B;n = 6–8) tests. Capsazepine (Cz) and 4-aminophenol were injected bythe intraperitoneal route 30 min and 10 min before the tests,respectively. *p,0.05, **p,0.01 and ***p,0.001 when compared tovehicle injection, using Mann-Whitney U test (A) or repeated measurestwo-way ANOVA followed by Sidak’s multiple comparisons test (B).doi:10.1371/journal.pone.0070690.g008

Figure 9. TRPV1 in brain mediates the antinociceptive effectsof 4-aminophenol and 4-hydroxy-3-methoxybenzylamine(HMBA). Intracerebroventricular injection of the TRPV1 blockercapsazepine (Cz) prevented the antinociceptive effect of 4-aminophe-nol (A; n = 5–7) and HMBA (B; n = 6–8) in the mouse formalin test. 4-aminophenol (30 mg/kg) and HMBA (100 mg/kg) were injected by theintraperitoneal route 10 min before the test. Capsazepine (100 nmol)was injected 5 min before the 4-aminophenol and HMBA administra-tion. **p,0.01 when compared to vehicle injection, using Mann-Whitney U test.doi:10.1371/journal.pone.0070690.g009



Materials and Methods

Ethics StatementThe animal procedures were approved by the animal ethics

committees in Auvergne, France (CEMEA Auvergne, No. CE3–

06) and Lund, Sweden (Malmo/Lunds djurforsoksetiska namnd,

No. M 111-10). The behavioural tests were conducted in

accordance with the official edict presented by the French

Figure 10. The antinociceptive effect of 4-aminophenol isdependent on cannabinoid CB1 receptors. The cannabinoid CB1receptor antagonist AM251 (3 mg/kg) prevented the antinociceptiveeffect of 4-aminophenol (100 mg/kg) in the rat formalin (A; n = 8–11)and paw pressure (B; n = 6–7) tests. However, 4-aminophenol (100 mg/kg) did not affect the global brain levels of the endocannabinoidsanandamide (C; n = 6) and 2-arachidonoylglycerol (2-AG; D; n = 6).AM251 and 4-aminophenol were injected by the intraperitoneal route30 min and 10 min before the behavioural assays, respectively. Thecontents of endocannabinoids are presented as normalized peak area(nPA) per g protein. *p,0.05, **p,0.01 and ***p,0.001 whencompared to vehicle injection, using Mann-Whitney U test (A), repeatedmeasures two-way ANOVA followed by Sidak’s multiple comparisonstest (B) or Kruskal-Wallis one-way ANOVA followed by Dunn’s multiplecomparisons test (C and D).doi:10.1371/journal.pone.0070690.g010

Figure 11. The antinociceptive effect of 4-aminophenol isdependent on spinal serotonergic mechanisms. Pre-treatment ofrats with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) reducedthe 4-aminophenol-induced increase of the withdrawal threshold in thepaw pressure test (A; n = 7–8). The serotonin receptor antagoniststropisetron (B; n = 7–8) and WAY-100635 (C; n = 6–9) prevented theantinociceptive effect of 4-aminophenol in the rat paw pressure andformalin tests, respectively. 5,7-DHT (100 mg) was administeredintrathecally 7 days before the paw pressure test. 4-Aminophenol(100 mg/kg) was injected by the intraperitoneal route 10 min beforethe behavioural tests. Tropisetron (Trop; 500 ng) and WAY-100635(WAY; 40 mg) were injected intrathecally 5 min before administration of



Ministry of Agriculture (Paris), the European Community Council

Directive (86/609/EEC) and the International Association for the

Study of Pain (IASP) guidelines for animal experiments [45].

AnimalsExperiments were performed on adult C57BL/6 mice (20–30 g)

and Sprague-Dawley rats (175–225 g) from Charles River

Laboratories (Lyon, France) or Taconic (Denmark). Only male

animals were used in the behavioral tests. TRPV12/2 and

FAAH2/2 mice were originally generated by David Julius [46]

and Benjamin Cravatt [47], respectively. These mice were back-

crossed with C57BL/6 mice for at least ten generations and their

genotype confirmed by polymerase chain reaction. Animals were

housed under controlled environmental conditions (21–22uC; 55%

humidity) and kept under a 12/12 h light/dark cycle with food

and water ad libitum for at least one week prior to the start the

experiments.

Assessment of TRPV1 ActivityTRPV1 activation was assessed indirectly on rat isolated

mesenteric arteries, using nerve-mediated vasorelaxation as

readout [2]. Briefly, arterial segments (2 mm long) were suspended

between two stainless steel wires in temperature-controlled tissue

baths (37uC), containing aerated (95% O2 and 5% CO2)

physiological salt solution (composition in mM: NaCl 119, KCl

4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 15, NaH2PO4 1.2 and D-

glucose 6; pH 7.4), under a passive load of 2 mN. One of the wires

was connected to a force-displacement transducer model FT03 C

(Grass Instruments; Rhode Island, USA) for isometric tension

recording. Vasorelaxation was studied in arterial segments

submaximally contracted with phenylephrine. Increasing concen-

trations of AM404, arvanil, olvanil and HPODA in the absence

and presence of capsazepine (3 mM) were then added cumulatively

to determine concentration-response relationships. All experi-

ments were performed in the presence of indomethacin (10 mM)

and Nv-nitro-L-arginine (300 mM) to reduce cyclooxygenase and

nitric oxide synthase activity, respectively. Under these conditions,

phenylephrine induces stable and long-lasting contractions, which

were minimally affected by the vehicle used.

4-aminophenol. *p,0.05, **p,0.01 and ***p,0.001 when compared tovehicle injection, using repeated measures two-way ANOVA followedby Sidak’s multiple comparisons test (A and B) or Mann-Whitney U test(C).doi:10.1371/journal.pone.0070690.g011

Figure 12. Proposed mechanism by which 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA) produce antinocicep-tion. Following passage through the blood-brain barrier, 4-aminophenol and HMBA are conjugated with free fatty acids (FFA), preferentiallyarachidonic acid, to yield the potent TRPV1 activators AM404 and arvanil plus olvanil, respectively, a reaction catalyzed by the enzyme fatty acidamide hydrolase (FAAH). AM404, arvanil and olvanil then activate TRPV1 in brain nuclei, regulating descending antinociceptive pathways, possibly ofserotoninergic origin, thereby reducing nociceptive neurotransmission in the dorsal horn of the spinal cord. In contrast to the other lipid metabolites,HPODA is produced by a FAAH-independent pathway and it does not seem to contribute to the antinociceptive action of 4-aminophenol, consistentwith HPODA being produced in smaller quantities and being a less potent TRPV1 activator than AM404.doi:10.1371/journal.pone.0070690.g012



Biochemical StudiesExperiments on brain homogenates in vitro. Rat brain

homogenate were prepared, as previously described [2]. Brains

were homogenized in Tris buffer (10 mM, pH 7.6), containing

ethylenediaminetetraacetic acid (EDTA; 1 mM), at volumes of 5–

10 ml/g tissue. Experiments were carried out on aliquots of 200 ml

homogenate at 37uC as further explained in the text. The

reactions were stopped by adding 1 ml ice-cold acetone, contain-

ing 1 mM [2H8]-anandamide. The samples were kept on ice until

the acetone phase was vacuum evaporated.

In vivo experiments. 4-Aminophenol (30 and 100 mg/kg)

and HMBA (100 and 300 mg/kg) were administered to mice and

rats by intraperitoneal injections in volumes of 10 ml/kg. Twenty

min after drug administration, the animals were anesthetized by

CO2 (rats) or isoflurane (mice) inhalation and decapitated. The

brain was removed from the skull and blood was collected in test

tubes, containing 60 ml buffered citrate, and the tissues snap frozen

in liquid nitrogen before storage at 270uC.

The whole brain was homogenized in ice-cold 10 mM Tris

buffer (ml/g tissue) at pH 7.6. The buffer solution contained

1 mM EDTA, 0.3 mM ascorbic acid and 10 mM methylarachi-

donylfluorophosphonate (MAFP) to prevent degradation of

AM404, arvanil, olvanil, anandamide and 2-arachidonoylglycerol.

Aliquots (200 ml) of blood and homogenates were precipitated with

1 ml ice-cold acetone, containing AM404-D4 (5 nM) or ananda-

mide-D8 (100 nM) as internal standard. After centrifugation at

25,200 g for 30 min at 4uC, the supernatants were collected in

polypropylene tubes and vacuum evaporated. The protein content

of the pellet was determined with Coomassie protein assay (Pierce;

Illinois, USA), using bovine serum albumin as standard. The

extraction residues were reconstituted in 100 ml methanol

(AM404, arvanil, olvanil anandamide and 2-arachidonoylglycerol)

or 0.5% acetic acid (4-aminophenol and HMBA). Samples were

analyzed on a Waters Aquity UPLC system coupled to a Sciex

5500 tandem mass spectrometer (LC-MS/MS; AB Sciex; Massa-

chusetts, USA) or a Perkin Elmer 200 HPLC system coupled to an

API 3000 tandem mass spectrometer (LC-MS-MS; Applied

Biosystems/MDS-SCIEX).

Mass spectrometry. Quantification of AM404, HPODA, arvanil

and olvanil. Samples were analyzed on a Waters Aquity UPLC

system coupled to a Sciex 5500 tandem mass spectrometer.

Sample aliquots of 5 ml were injected into an Acquity UPLC BEH

C18 column (100 6 2 mm, 1.7 mm; Waters) held at 50uC. All

mobile phases were water-acetonitrile gradients, containing 0.1%

formic acid and using a flow rate of 400 ml/min. The acetonitrile

content was initially 30%, then increased linearly to 100% over

7 min and kept at this level for 4 min, followed by an equilibration

period at 30% for 2 min. The electrospray interface was operating

in the positive ion mode at 550uC and the ion spray voltage was set

to 5000 volts. The mass transitions (m/z) used were the following:

396.2/110.1 for AM404, 374.2/110.0 for HPODA, 400.2/114.0

for AM404-D4, 440.3/137.1 for arvanil and 418.3/137.1 for

olvanil. The declustering potentials were between 96 and 130 volts

and the collision energies between 25 and 41 volts.

Quantification of 4-aminophenol and HMBA. Sample aliquots of 5 ml

were injected into a Hypercarb column (50 6 2.1 mm; Thermo).

All mobile phases were water-acetonitrile gradients, containing

0.1% formic acid. The acetonitrile content was initially 2%, then

increased linearly to 75% over 5 min and then kept at 100% for

1 min, followed by an equilibration period at 2% for 1.5 min. The

electrospray ion source was set at 550uC and used in the positive

ion mode with an ion spray voltage of 5000. The mass transitions

(m/z) was 110.1/93.0 for 4-aminophenol. HMBA loses the amino

group already in the source of the MS and therefore the mass

transition used was 137.0/94.0. The declustering potentials were

100 and 90 volts and the collision energies 21 and 29 volts. No

internal standard was used for these analyses.

Quantification of anandamide and 2-arachidonoylglycerol. Anandamide

(m/z 348.2/62.0), anandamide-D8 (m/z 356.4/63.0) and 2-

arachidonoylglycerol (m/z 379.2/287.0) were determined as

previously described [3]. Since 2-arachidonoylglycerol is non-

enzymatically converted to 1-arachidonoylglycerol, the content of

2-arachidonoylglycerol was estimated as the sum of these lipids.

The contents of the compounds were expressed either in moles

or as normalized peak areas (nPA) and related to the protein

content in the samples. The nPA was obtained by dividing the

peak area of the analyte with the peak area for the internal

standard in the same sample. The detection limits were calculated

as the concentrations corresponding to three times the standard

deviation of the blanks. When levels were below detection limit,

numerical values of half the detection limit were used in the

calculations.

Behavioural TestsThe behavioural tests were performed in a quiet room and

evaluated by a single investigator in a blinded manner. Treatments

and/or genotypes were randomized in blocks and the number of

animals in each block corresponded to the number of treatments

and/or genotypes compared. For example, when the effect of

capsazepine on the antinociceptive responses to 4-aminophenol

was tested, four animals were included in each block, correspond-

ing to the following treatments: Vehicle+vehicle, vehicle +4-

aminophenol, capsazepine+vehicle and capsazepine +4-amino-

phenol. Each animal was exposed to only one treatment. All

behavioral tests except the rat paw pressure test were performed

10 min after the intraperitoneal injection of 4-aminophenol,

HMBA, dipyrone or vehicle. PMSF (s.c.) and capsazepine (i.p.)

were administrated 20 min and 10 min before 4-aminophenol

(i.p.), respectively. Capsazepine (i.c.v.), tropisetron (i.t.) and WAY-

100635 (i.t.) were injected 5 min before 4-aminophenol (i.p.) or

HMBA (i.p.).

Assessment of locomotor activity – mouse. Mice were

placed in actimetry boxes (Actisystem; Apelex, France) and

spontaneous motor activity was assessed by determining the

number of crossings of light beams during 15 min.

Formalin test – mouse and rat. The animals received an

intraplantar injection of a 2.5% formalin solution (50 ml rat, 25 ml

mouse) into a hind paw. The time spent biting and licking of the

injected paw was monitored during the two typical phases of the

nociceptive response (phase I: 0–5 min; phase II: 20–40 min in

rats or 15–40 min in mice).

Paw pressure test – rat. The rats were submitted to the paw

pressure test, using an Ugo Basile analgesy meter with a probe tip

diameter of 1 mm (Bioseb, France). Nociceptive thresholds,

expressed in grams (g), were measured by applying an increasing

pressure to the right hind paw until a squeak (vocalization

thresholds) was obtained (cut-off 750 g). Two consecutive stable

vocalisation threshold values were first obtained (baseline). The

various treatments were then given and the measurements

repeated 15, 30, 45, 60 and 90 after 4-aminophenol or vehicle

administration.

von Frey filament test – mouse. Calibrated von Frey

filaments (0.0045–5.4950 g) were used to induce light noxious

mechanical stimulation in mice [48]. Tests were commenced after

one hour of habituation. Filaments of increasing stiffness were

applied five times perpendicular to the plantar surface of the

hindpaw and pressed until bending. The first filament that evoked



at least three consecutive responses was assigned as the threshold

(cut-off 2 g).Tail immersion test – mouse. Tails of mice were

submerged in a hot water bath (46uC) and the withdrawal latency

recorded (cut-off 15 s). The mean of four baseline values were

determined before drug administration.Intracerebroventricular (mouse) and intrathecal (rat)

injections. Capsazepine (100 nmol) and methylene blue

(20 mg) were injected into the lateral ventricle of mice, using a

25 ml Hamilton syringe. The injection site was 1 mm lateral to the

midline drawn through the anterior base of the ears. The syringe

was inserted perpendicularly through the skull into the brain to a

depth of 4 mm, where 2 ml of the solution was injected, as

previously described [3]. Mice were sacrificed 45 min after

methylene blue injection and the brain and the thoracic and

lumbar spinal cord were removed for ocular inspection. Intrathe-

cal injections were performed on anaesthetised rats held in one

hand by the pelvic girdle. A 25 gauge 1 inch needle connected to a

25 ml Hamilton syringe was then inserted into the subarachnoidal

space between lumbar vertebrae L5 and L6 until a tail flick was

elicited. The syringe was held in position for few seconds after the

injection of a volume of 10 ml [49]. All injections were performed

under isoflurane anaesthesia (4% induction, 2% maintenance).Lesion of the descending serotonergic pathways –

rat. 5,7-Dihydroxytryptamine (5,7-DHT; 100 mg/rat) was ad-

ministered intrathecally 7 days before the experiment. Desipra-

mine (10 mg/kg) was administered (i.p.) 30 min before 5,7-DHT

to prevent the uptake of 5,7-DHT by monoaminergic neurons.

After the experiment, the animal was sacrificed and the lumbar

spinal cords collected for quantification of serotonin by HPLC

[12]. Briefly, the dorsal horn of the lumbar spinal cord was

homogenized in a saturated KCl solution and centrifuged at

15,000 g for 15 min. The resulting supernatants were mixed with

400 ml of an internal standard (n-methyl-serotonin 0.6 mg/l,

diluted in glycine buffer at pH 11) and extracted with 2.5 ml of

dichloromethane/butanol (75/25). The organic layer was back-

extracted with 300 ml of 0.1 M phosphate buffer (pH 4.4). One-

hundred ml of this solution was injected in the HPLC system with

an electrochemical detector.

Drugs4-Aminophenol, 4-hydroxy-3-methoxybenzylamine (HMBA),

phenylmethylsulfonyl fluoride (PMSF), N-[2-(4-Chloropheny-

l)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-

carbothioamide (capsazepine), N-[2-[4-(2-methoxyphenyl)-1-piper-

azinyl]ethyl]-N-2-pyridinylcyclohexane-carboxamide maleate

(WAY-100635), 5,7-dihydroxytryptamine (5,7-DHT) and desipra-

mine were purchased from Sigma-Aldrich (France). N-(4-Hydro-

xyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), N-(4-hy-

droxy-3-methoxybenyl)-9Z-octadecenamide (olvanil),

anandamide, 2-arachidonoylglycerol and N-(piperidin-1-yl)-5-(4-

iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-car-

boxamide (AM251) were obtained from Tocris Cookson (UK). N-

(4-hydroxy-3-methoxybenzyl)-5Z,8Z,11Z,14Z-eicosatetraenamide

(arvanil) was purchased from Cayman Chemicals (USA). Tropise-

tron was a gift from Novartis (France) and N-(4-hydroxyphenyl)-

9Z-octadecenamide (HPODA) was provided by Prof. Olov

Sterner (Lund University, Lund, Sweden). AM404, arvanil,

olvanil, and HPODA were dissolved in ethanol. PMSF and

AM251 were dissolved in DMSO. Capsazepine was dissolved in

ethanol (in vitro tests), DMSO (intraperitoneal injection) or

physiological serum/DMSO/Tween 80 (85/10/5; intracerebro-

ventricular injection). 4-Aminophenol was dissolved in physiolog-

ical saline (mouse) or DMSO (rat). HMBA, tropisetron, 5,7-DHT

and desipramine were dissolved in physiological saline (0.9%

NaCl). The 5,7-DHT solution contained 0.2 mg/ml ascorbic acid.

Intraperitoneal and subcutaneous injections were given in volumes

of 5–10 ml/kg.

Calculations and Statistical AnalysisData are presented as the mean 6 SEM, and n indicates the

number of animal tested in each group. pEC50 of the lipid

metabolites indicates the negative log concentration that elicited

half-maximal vasorelaxation. Mann-Whitney U test or Student’s t-

test was used when two groups were compared. When multiple

groups were compared, Kruskal-Wallis one-way ANOVA fol-

lowed by Dunn’s multiple comparisons test was used. Data from

the paw pressure test were analyzed by repeated measures two-

way ANOVA followed by Sidak’s or Dunnett’s multiple compar-

isons test. The level of statistical significance was set at p,0.05.

GraphPad Prism 6.0 software (California, USA) was used in all

calculations and statistical analyses.

Supporting Information

Figure S1 Arvanil, olvanil and N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) produce TRPV1-dependentvasorelaxation. The TRPV1 blocker capsazepine significantly

suppressed the vasorelaxation evoked by arvanil (p,0.01), olvanil

(p,0.01) and HPODA (p,0.001) in rat mesenteric arterial

segments precontracted with phenylephrine. Unfilled and filled

circles indicate vasodilator responses in the absence (n = 7–12) and

presence (n = 6–7) of capsazepine (3 mM), respectively (the former

values are the same as in Fig. 1B). Filled triangles indicate

responses after pretreatment (60 min) of the vascular segment with

capsaicin (1 mM) to inactivate capsaicin-sensitive nerve fibres

(n = 6–8). Values are expressed as mean 6 SE. Mann-Whitney U

test was used to compare the areas under the concentration-

response curve in the absence and presence of capsazepine.

Sigmoidal concentration-response curves (variable slope) were

constructed, using GraphPad Prism 6.0 software (California,

USA).

(TIF)

Acknowledgments

N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) was kindly provided

by Prof. Olov Sterner.

Author Contributions

Conceived and designed the experiments: AE EDH PMZ. Performed the

experiments: CM CS DAB EC EDH FL LD ME PMZ. Analyzed the data:

AB AE CM DAB EDH PMZ. Wrote the paper: AE CM EDH PMZ.

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